Without limiting the scope of the invention, its background is described in connection with existing apparatus and methods for use in combinatorial chemistry using split synthesis. Combinatorial chemistry refers to the production of libraries of diverse compounds that have the same constituent elements but are assembled in different sequence through the repetitive sequential covalent addition of the constituent subunits. Combinatorial chemistry is able to generate large populations of random sequence polymers, especially random sequence short chain polynucleotide and polypeptide polymers. Gordon, E. M., et al., J. Med. Chem. 37 (1994) 1385.
Combinatorial libraries are created by one of three general methods—parallel synthesis, one pot synthesis, or split synthesis. In parallel synthesis, different compounds are synthesized in separate vessels either on a solid support or in solution. A commonly used format for parallel synthesis in fixed arrays coupled with robotics instrumentation to add different reagents to separate wells of the array in a predefined manner to produce combinatorial libraries where the molecular structure of each product is defined by its location in the array. Fodor et al., Science 251 (1991) 767; U.S. Pat. No. 5,143,854; WO 93/09668. For example, the BioAutomation MERMADE instruments (Plano, Tex.) provide a 96 well format that can be used for parallel synthesis with tracking provided by XY table addressing of individual wells. When each individual member of the array is tested for activity, the composition of positive members of the array is known by review of the tracking and can be resynthesized. Parallel synthesis of this type yields relatively large quantities of a relatively small number of candidate compounds. Alternatively, libraries can be synthesized in arrays on microchips followed by simultaneous assay for binding or activity. Using photolithography, AFFYMAX (Palo Alto, Calif.) has generated arrays of more than 65,000 compounds on chips of about 1 square centimeter in area. Development of improved parallel synthesis methods and apparatus has been a primary focus of the combinatorial chemistry field. However, parallel synthesis remains limited by the number of individual molecules that can be generated depending on the size of the array.
In one pot synthesis, combinatorial libraries are made using mixtures of compounds added at each coupling step. However, in a coupling step, mixed reagents can compete to become integrated at the same site resulting in unequal distribution of components. Because the widely different coupling rates of different activated amino acids leads to unequal representation, split synthesis using solid particulate supports was developed for the generation of peptide libraries. Furka et al. Int. J. Pept. Protein Res. 37 (1991) 487; Lam, et al. Nature 354 (1991) 82 and WO92/00091, U.S. Pat. Nos. 5,650,489 and 5,651,943; Houghten et al., Nature 354 (1991) 84 and WO 92/09300; Hueber and Santi, U.S. Pat. No. 5,182,366. In addition, Lam et al. developed methods of screening one-bead one-compound (OBOC) libraries of peptides where each bead presents a single peptide species. Lam et al. supra.
In split synthesis, a plurality of base moieties are split into separate reaction vessels each for addition of a different further moiety. After addition, the groups are pooled and again split into the separate reaction vessels for addition of the next building block via a second round of derivatization and so on. Where particulate solid supports such as derivatized “beads” are used, the bound products of each cycle are retained on the solid phase while excess reagents and byproducts are washed away. By successive treatment with different reagents and/or addition of different chemical elements, the bound products are elongated on the solid support to form a large library of related compounds that are tested for desired properties. However, the loading capacity of beaded supports and the resulting low yield of each product for testing purposes has limited the applicability of split synthesis technologies and, consequently, pharmaceutical companies have preferred to use and further develop automated parallel synthesis methods for preparation of combinatorial libraries. See Lam et al. WO04/085049; Furka U.S. Pat. No. 6,420,123.
Candidate libraries of random sequence oligonucleotides can be generated manually or by modifying the normal operation of sequence specific nucleic acid synthesizers. However, commercial nucleic acid synthesizers are specifically designed to produce defined sequence oligonucleotides by the sequential programmed stepwise addition and coupling of single selected nucleotide bases. In order to generate random nucleic acid oligomers, commercially available nucleic acid synthesizers have been adapted to perform “mixed” synthesis. This essentially constitutes “one-pot” synthesis in which a mixture of nucleotide bases is added in lieu of a single defined base at each coupling step. Using mixed one-pot synthesis, each bead contains a number of different oligonucleotide species which are later cleaved from the support. This adaptation does not permit compensation for differing reaction rates and may result in skewing of the resultant population.
Split synthesis as originally adapted to generation of single bead peptide libraries has been recently applied to generate one-bead one-oligonucleotide libraries where each bead presents many copies of a single oligonucleotide sequence or species. However, available DNA synthesizers are not designed for split synthesis. Thus, the products from each round of synthesis must be manually pooled and split for subsequent rounds of synthesis, thus limiting the productivity of the synthesis.
From the foregoing it is apparent the there is a need in the art for apparatus and methods that are able to provide for the generation of representational libraries by split combinatorial synthesis, including the efficient automation of such processes.